Objective:To describe our novel modification in isolating pancreatic stellate cells (PSCs) from normal human pancreas and human pancreatic ductal adenocarcinoma (PDAC) tissue. Methods:Normal PSCs were isolated with enzyme digestion and ladder centrifuge. Isolated PSCs were cultured in DMEM/F12 containing 10% fetal bovine serum. Cancer-associated PSCs were obtained by an outgrowth method. Isolated PSCs were cultured in DMEM/F12 containing 20% fetal bovine serum. Results:With our modifications,normal pancreas tissue from human yields an adequate number of PSCs (approximately 0.5-5 million/g pancreas) for in vitro studies,and the cell viability was about 90%. After new outgrowth method applied,tissue blocks were attached more tightly and cells grew out earlier compared to the previous method. Primary isolated PSCs were verified with appearance,auto-fluorescence,positive expression of α-SMA,Vimentin,Desmin,GFAP. Conclusion:Our modification for PSCs isolation significantly increase the isolating efficiency with shorter culture period,which can provide great convenience for future researches on PSCs.